Given the role of the peptides of the invention in modulating the activity of cellular protein phosphatase 2A, it is important in the introduction to recall the current knowledge regarding protein phosphatase 2As, their physiological role and their interactions with certain cellular, viral or parasitic proteins.
Cell physiology is partially controlled by modulating protein phosphorylation. The phosphorylation state of cell proteins depends on the antagonist action of protein kinases which phosphorylates them and protein phosphatases which dephosphorylate them.
Protein phosphatases are divided into two principal groups: tyrosine phosphatases and serine/threonine phosphatases. Serine/threonine phosphatases are classified into two categories which depend on the specificity of their substrate and their sensitivity to certain inhibitors, namely type 1 phosphatases (PP1) and type 2 phosphatases (PP2). Type 2 phosphatases are themselves divided into different classes, including phosphatase 2A (PP2A), phosphatase 2B or calcineurine the activity of which is regulated by calcium, and phosphatase 2C (PP2C) the activity of which is regulated by magnesium.
It is now known that type 2A phosphatases are highly conserved during evolution and are potentially involved in regulating many biological processes. PP2A enzymes have been clearly involved in regulating transcription, control of the cell cycle or viral transformation. Further, PP2As are targeted by different viral or parasitic proteins, suggesting a role for PP2As in host-pathogen interactions.
PP2As are oligomeric complexes (holoenzymes) each comprising a catalytic subunit (C) and one or two regulating subunits (A) and (B). The structure of subunit (A) consists of 15 imperfect repeats of a conserved amino acid sequence of 38 to 40 amino acids, certain of which interact with subunits (B) and (C). Subunits (A) and (C), conserved during evolution, constitute the base structure of the enzyme and are expressed constitutively. In contrast, subunits (B) constitute a family of regulating proteins not connected via a common structure and expressed differentially (Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem 1989; 58: 453-508). Protein phosphatase 2As exist in vivo in two classes with different forms: a dimeric form (AC) and a trimeric form (ABC). Subunits (B) regulate phosphatase activity and specificity towards the substrate. The existence of multiple forms of PP2A is correlated with the distinct and varied functions of PP2A in vivo.
Recently, different proteins synthesized by pathogens, in particular viral and parasitic proteins, have been implicated in modulating certain specific activities of protein phosphatase 2A.
Different strategies involving PP2A have been adopted by viruses to facilitate their replication and survival in a host cell. As an example, parainfluenza virus incorporates the protein PKCζ, a protein of cellular origin under the control of PP2A, into its viral particle. This can perturb the phosphoylation of host proteins and facilitate its own replication (De B P, Gupta S, Barnejee A K. Cellular protein kinase C ζ regulates human parainfluenza virus type 3 replication. Proc. Natl. Acad Sci USA 1995; 92: 5204-8).
Several DNA viruses with transforming power, such as papovae or adenoviruses, as well as certain retroviruses such as the type 1 human immunodeficiency virus (HIV-1), code for proteins which interact directly with certain host PP2As. All of those viruses comprise proteins which, although structurally different, interact with certain holoenzymes and modify phosphatase activity.
In particular, it has been shown that the E4orf4 protein of adenoviruses binds to a heterotrimeric PP2A and more precisely to a regulating subunit (B), which causes a reduction in the transcription of JunB in the infected cell. That effect could play an important role during viral infection by regulating the apoptotic response of infected cells. Interestingly, it has also been shown that the interaction of E4orf4 with PP2A induces apoptosis in transformed cells in a p53-independent manner (Shtrichman R et al, Adenovirus type 5 E4 open reading frame 4 protein induces apoptosis in transformed cells. J Virol 1998; 72: 2975-82).
Tumor-generating viruses of the Papovae family, including SV40 and polyoma virus, induce cell transformation. It has been shown that PP2A interacts with the “small T” antigen of SV40 or polyoma and with the transforming “middle T” protein of polyoma. Those interactions of viral proteins with PP2A have been clearly involved in viral transformation. Finally, transcriptional regulation, a process normally carried out in the cell by different factors specifically fixing to promoter regulating sequences, probably represents the most important mechanism involved in the control of viral expression by PP2A. It has been demonstrated that PP2A is a negative regulator for numerous transcription factors involved in particular in the processes of cell growth and proliferation, including AP1/SRE, NF-κB, Sp1 and CREB (Waszinski, B E, Wheat W H, Jaspers S, Peruski L F, J R Lickteig R L, Johnson G L, and Klemm D J, Nuclear protein phosphatase 2A dephosphorylates protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. Mol Cell Biol 1993 13, 2822-34). Viral regulation of those transcription factors would permit modulation of viral transcription.
The viral protein of HIV-1, Vpr, interacts in vitro with PP2A and stimulates the catalytic activity of PP2A (Tung L et al, Direct activation of protein phosphatase 2A0 by HIV-1 encoded protein complex Ncp7: vpr. FEBS Lett 1997; 401: 197-201). Vpr can induce the G2 stoppage of infected cells by inhibiting the activation of the p34cdc2-cycline B complex. Further, Vpr interacts with the transcription factor Sp1 and is a weak trans-activator for transcription of Sp1 dependent HIV-1. Thus, the Vpr protein of HIV-1, which is incorporated into the virion, should be involved in vivo in the initiation of viral transcription, a step that is clearly essential for regulating the expression of the Tat transcription factor (a major regulator of transcription encoded by the HIV-1 virus).
In contrast to the well established role of protein kinases in parasitic infections, it is only during the past three years that serine/threonine phosphatases have begun to be recognized as being important potential regulators in the field of parasitology.
Initially, two serine/threonine phosphatases, Ppβ and PfPP, were identified in Plasmodium falciparum. The presence of type 1 and type 2A phosphatase activity in the parasite has been demonstrated by enzymological studies. Finally, parasitic enzymes PP2A and PP2B were purified.
Serine/threonine phosphatases have recently been studied in Theileria parva, another protozoan close to P. falciparum, a cattle parasite. Monocyte and leukocyte host cells infected by the parasite are transformed, resulting in leukemia in the animal. Purified parasites of cells infected with Theileria express a protein kinase CK2α. Now, the subunit CK2α should interact with PP2A to positively modulate its activity (HérichéH, et al, Regulation of protein phosphatase 2A by direct interaction with casein kinase 2α. Science 1997; 276: 952-5). Further, modulation of PP2A via expression of the CK2α subunit could be the basis of blockage of two signal routes in the parasitised cell, that of MAP-kinases (Chaussepied M et al. Theileria transformation of bovine leukocytes: a parasite model for the study of lymphoproliferation. Res Immunol 1996; 147: 127-38) and that of protein kinase B (Akt) (M Baumgartner, M Chaussepied, M F Moreau, A Garcia, G Langsley. Constitutive PI3-K activity is essential for proliferation, but not survival, of Theileria parva—transformed B cells. Cellular Microbiol (2000) 2, 329-339).
The absence of common motifs to the series of proteins interacting with PP2A prevents the informatical identification of peptide motifs directly involved in binding those proteins with PP2A.
Given the major role of protein phosphatase 2As in virus-host interactions or parasite-host interactions as summarized above, the importance of identifying the binding sites of viral or parasitic proteins with PP2A holoenzymes or one of their subunits can be understood, so that novel therapeutic targets for those viral or parasitic pathogens can be identified.
In particular, the identification of peptides interacting with PP2A should allow novel drugs to be developed that can block, by competitive inhibition, the cell mechanisms induced by viral or parasitic proteins via their interaction with PP2A and in particular mechanisms of infection, pathogen proliferation and cell transformation.